LAMININ GENE EXPRESSION IN GLOMERULAR CELLS

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DESCRIPTION (Adapted from Investigator's Abstract): This is an amended application for competitive renewal, submitted by an experienced and highly-respected investigator. The LAMC1 gene encodes laminin gamma1 (formerly laminin B2) that is a component of heterotrimeric laminin, a molecule that is present in normal basement membranes and accumulates in glomerulosclerosis. In past work, the PI has shown that IL-1 and TGF- b, and PMA activate laminin gamma1 gene expression in rat glomerular epithelial cells. He has shown that NF-kB contributes to this process and has recently extended this line of work to a novel transcriptional factor. In comparing the 5' genomic sequence of human, rat, and mouse laminin gamma1 he discovered a novel and conserved sequence, termed bcn-1. He has shown that this sequence binds a nuclear factor or factors which are upregulated by PMA or IL-1b treatment of mesangial cells. The bcn-1 sequence is present in other promoters (complement, insulin) and the BCN-1 nuclear factor is also present in monkey kidney COS-7 cells and human Jurkat cells. He proposes three aims: 1. To characterize transcription factors which bind to the bcn-1 motif. Using the yeast one hybrid system, he has identified several factors (RTEF/EFT, TEF-3, and two partial novel cDNAs, K53 and K46). In yeast, K53 is of particular interest because it binds to an intact but not mutated bcn-1 sequence (unlike RTEF and K46 which bind both). In mesangial cells, both TEF3 and K46 activate the LAMC1 promoter with an intact bcn-1 site but not with a mutated bcn-1 sequence (the full cDNA for K53 is not available). Since BCN-1 activity may reside within a complex of which the individual components lack activity, the yeast hybrid system may give misleading results. He will therefore pursue a parallel strategy to fractionate nuclear extracts on an affinity DNA column and carry out microsequencing. Characterization of BCN-1 protein will include antibody production, comparison of size between recombinant product and nuclear extract protein on gel shift analysis, analysis of domains for DNA binding, transcriptional activation, and nuclear localization signal. 2. To identify LAMC1 intron elements and factors that are responsive to treatment of glomerular cells with IL-1b, TGF-b, and PMA. The rationale for these studies is data from the PI's laboratory that agonist stimulation of mRNA expression is greater than what can be shown due to agonist effect on the promoter. While acknowledging that increased message stability could account for these observations, he proposes that additional cis acting regulatory elements may be located in the first intron of the rat LAMC1 gene. He will therefore search for DNase hypersensitive sites in the rat gene. If the sites appear similar to those already published by Philips and colleagues in the mouse intron, he will use mouse clones provided by Dr. Phillips that boost gene transcription 20 fold, and test them in mouse glomerular cells. He will then search for sites in the data base and carry out deletion analysis, seek evidence from gel shift that known transcription factors bind to these sites, and determine whether the enhancer synergizes with the promoter. 3. To define transcriptional silencing elements in the LAMC1 5'-flanking region. In preliminary work, the PI has found suggesting a silencer in region -233/-15. He will attempt further localize the effect, carry out in vitro DNase footprinting, and search with the identified sequence in transcription factor databases. If the silencer can been narrowed down to 10-20 bp, then mutations can be made in the context of the -233/-15 fragment to abolish silencer activity. He will also test the silencer effect on heterologous promoters (SV40).